THE STOICHIOMETRY OF PHOTOPHOSPHORYLATION 



Thomas Punnett 



When non-cyclic photophosphorylation was first described by Arnon et al (1), 

 the ratio of ATP formed to electrons transported was found to be one. This 

 determination was repeated by Avron and Jagendorf (2), by Davenport (3), by 

 Good (4), by Stiller and Vennesland (5), by Turner et al (6) and others. In 

 all cases, the ATP/2e- ratio has been the same. 



There is some ambiguity in this determination, however, because of the 

 nature of the data (2). In a typical experiment, one obtains the rate of 

 phosphorylation, the rate of oxidant reduction in the presence of phosphate 

 acceptor and the rate of reduction in the absence of acceptor. The ATP/2e- 

 ratio can be calculated either by dividing the rate of phosphorylation by the 

 maximum rate of reduction, or by "correcting" the ADP-enhanced reduction rate 

 for the reduction occurring in the absence of ADP and dividing this "corrected" 

 value into the rate of phosphorylation. The usual effect of making this 

 correction is to increase the ATP/2e- ratio from one to two or three. Arnon 

 and Avron and Jagendorf elected to use the more conservative uncorrected value 

 of one while Good has argued in favor of the corrected value of two. Stiller 

 and Vennesland, in their earlier study only, found a good stoichiometric 

 relation but concluded that this relation was fortuitous and that the two 

 processes were not related to each other in a quantitative manner. We have 

 held both of these latter two views at various stages of this investigation. 



The first problem, therefore, was that of the treatment of the date. Our 

 approach to this problem was to study reaction rates as a function of several 

 variables to see whether a constant stoichiometry could be obtained . The 

 basic assumption was that if phosphorylation and reduction were the conse- 

 quence of a single process, then the overall rates should vary with pH, 

 concentration of oxidant, etc., but the stoichiometry should be constant. 

 Furthermore, a constant stoichiometry should be found only with one of the 

 two methods of treating the data. 



FERRICYANIDE PHOSPHORYLATION 



In these experiments, done with the collaboration of Dr. Rajul V. Iyer, we 

 used oat chloroplasts (Avena sativa, var . Garry) at a concentration of 7 to 

 20 microgramsj^er ml., high intensity red light and one to two minute reaction 

 times. In the determinations of pH curves for the ferricyanide Hill reaction, 

 we found that the addition of ADP had no effect on the reduction rates between 

 pH 6.8 and 7.5 although there was an appreciable rate of phosphorylation. 

 When these data were used to calculate an ATP/2e- ratio, the "corrected" ratio 

 was infinite. It decreased to 2 only when the pH was increased to 7.9 and 

 was variable with further increase to pH 8.7. This response, and that des- 

 cribed below in the quinone Hill reaction, lead us to conclude that it is 

 improper to "correct" the reduction rates. 



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